LOCALIZATION OF CYS(133) OF RABBIT SKELETAL TROPONIN-L WITH RESPECT TO TROPONIN-C BY RESONANCE ENERGY-TRANSFER

We have used the technique of resonance energy transfer in conjunction with distance geometry analysis to localize Cys(133) of troponin-l (T nl) with respect to troponin-C (TnC) in the ternary troponin complex a nd the binary TnC.Tnl complex in the presence and absence of Ca2+. Cys (133) of Tnl was chosen because our previous work has shown that the r egion of Tnl containing this residue undergoes Ca2+-dependent movement s between actin and TnC, and may play an important role in the regulat ory function of troponin. For this purpose, a Tnl mutant with a single Cys at position 133, and TnC mutants, each with a single Cys at posit ions 5, 12, 21, 41, 49, 89, 98, 133, and 158, were constructed by site -directed mutagenesis. The distances between Tnl Cys(133) and each of the nine residues in TnC were then measured. Using a least-squares min imization procedure, we determined the position of Tnl Cys(133) in the coordinate system of the crystal structure of TnC. Our results show t hat in the presence of Ca2+, Tnl Cys(133) is located near residue 12 b eneath the N-terminal lobe of TnC, and moves away by 12.6 Angstrom upo n the removal of Ca2+. Tnl Cys(133) and the region of TnC that undergo es major change in conformation in response to Ca2+ are located roughl y on opposite sides of TnC's central helix. This suggests that the reg ion in Tnl that undergoes Ca2+-dependent interaction with TnC is disti nct from that interacting with actin.